The plasma membranes of many vertebrate cells contain integral proteins, called Na+/H+ exchangers, which are involved in regulation of intracellular pH, cell volume, and growth. A Na+/H+ exchange,, referred to as the "housekeeping"-type, is present in many cells including the basolateral membrane of epithelia lining the mammalian nephron. Another Na+/H+ exchanger with different physiological properties, the "epithelial"type, is present on the apical membrane where it mediates active transport of NaHCO3 and NaCl. Abnormalities of these Na+/H+ exchangers may be involved in the pathogenesis of metabolic acidosis, edemaformation, and abnormal growth. The overall aim of this project is to clone and characterize the genes encoding rabbit Na+/H+ exchangers and to study the regulated expression of the genes in the kidney. Recently, transcripts (cDNAs) encoding human, rabbit, and porcine "housekeeping"-type Na+/H+ exchangers have been cloned. We have used these cDNAs to isolate 17 kb of the corresponding rabbit Na+/H+ exchanger gene. The 17-kb genomic clone contains sequences upstream of the transcript (5' flanking sequence) where the gene promoter is located. We will characterize the structures of the 17-kb and other genomic clones, including locations of intron-exon junctions and transcription initiation sites. We have also shown that steady-state levels of mRNA encoding the "housekeeping"-type Na+/H4+ exchanger in porcine renal cells (LLC-PKI) are increased following acidification of the medium. This is consistent with transcriptional stimulation of the Na4+/H+ exchanger gene in response to metabolic acidosis. The proposed studies will examine in greater detail the time course and dose-response of these effects. We will evaluate the effects of acidification on rates of transcription, mRNA stability, and transcript processing. To define the promoter of the "housekeeping"-type Na+/H+ exchanger gene, portions of the 17-kb genomic clone containing 5' flanking sequence will be ligated to a reporter gene, luciferase, and transiently expressed in cultured cells to assess promoter strength. Gene enhancers or silencers will be identified by their effects on a heterologous promoter. Since the "housekeeping"-" Na+/H+ exchanger is only present in certain nephron segments, studies employing cultured cells or transgenic mice will examine the basis for tissue-specificity of gene expression. The cis-elements and transcription factors responsible for responsiveness to metabolic acidosis and tissue-specific expression will be identified. When cDNAs encoding the "epithelial"-type Na+/H+ exchanger become available, a similar analysis of its gene will be performed. The proposed studies of Na+/H+ exchangers are relevant to understanding the physiology and pathophysiology of membrane transport. Such studies will clarify the mechanisms underlying the renal and cellular responses to metabolic acidosis. Moreover, characterizing the genes encoding Na+/H+ exchangers and identifying their promoters, regulatory elements, and transcription factors will contribute to our overall understanding of eukaryotic gene expression.